Abstract
Fungal effector proteins facilitate host‐plant colonization and have generally been characterized as small secreted proteins (SSPs). We classified and functionally tested SSPs from the secretomes of three closely related necrotrophic phytopathogens: Ciborinia camelliae, Botrytis cinerea, and Sclerotinia sclerotiorum. Alignment of predicted SSPs identified a large protein family that share greater than 41% amino acid identity and that have key characteristics of previously described microbe‐associated molecular patterns (MAMPs). Strikingly, 73 of the 75 SSP family members were predicted within the secretome of the host‐specialist C. camelliae with single‐copy homologs identified in the secretomes of the host generalists S. sclerotiorum and B. cinerea. To explore the potential function of this family of SSPs, 10 of the 73 C. camelliae proteins, together with the single‐copy homologs from S. sclerotiorum (SsSSP3) and B. cinerea (BcSSP2), were cloned and expressed as recombinant proteins. Infiltration of SsSSP3 and BcSSP2 into host tissue induced rapid necrosis. In contrast, only one of the 10 tested C. camelliae SSPs was able to induce a limited amount of necrosis. Analysis of chimeric proteins consisting of domains from both a necrosis‐inducing and a non‐necrosis‐inducing SSP demonstrated that the C‐terminus of the S. sclerotiorum SSP is essential for necrosis‐inducing function. Deletion of the BcSSP2 homolog from B. cinerea did not affect growth or pathogenesis. Thus, this research uncovered a family of highly conserved SSPs present in diverse ascomycetes that exhibit contrasting necrosis‐inducing functions.
Highlights
Some of the most economically important eukaryotic phytopathogens are fungi (Dean et al, 2012)
Fungal secretome prediction is a popular strategy for identifying putative effectors that are required for fungal virulence or pathogenicity (Amselem et al, 2011; Hacquard et al, 2012; Morais do Amaral et al, 2012)
Recombinant protein assays indicated that several members of the CCL-small secreted proteins (SSPs) family (BcSSP2 and SsSSP3) could induce host-cell necrosis and that the C-terminal half of SsSSP3 was essential for this necrosis-inducing function
Summary
Some of the most economically important eukaryotic phytopathogens are fungi (Dean et al, 2012). By screening fungal secretomes for conserved small secreted proteins (SSPs), it may be possible to select for proteins that induce host-cell necrosis To test this hypothesis, we predicted and compared the secretomes of the three necrotrophic fungal phytopathogens Botrytis cinerea, Sclerotinia sclerotiorum, and Ciborinia camelliae. Nine of the 10 culture filtrates that contained C. camelliae CCL-SSP protein homologs failed to stimulate a visible host response by 24 hr post-infiltration (Figures 3a and S5a). Of the eight chimeric proteins infiltrated into petal tissue, only CcSSP37 substituted with the exon 2 region of SsSSP3 (SubsSsSSP3 Ex2) was able to induce strong necrosis, suggesting that the region encoded by exon 1 is not responsible for necrosis-inducing activity These results align with the hypothesis that the N-terminal region is predominantly a signal peptide. These results indicate that BcSSP2 is not essential for B. cinerea virulence
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